Cytokine regulation of cannabinoid receptors 1 & 2 in multiple sclerosis

Lucie Jean-Gilles, Cris S. Constantinescu. University of Nottingham, Nottingham, United Kingdom.

Multiple Sclerosis (MS) is an inflammatory disease of CNS white matter which is characterized by focal T cell and macrophage infiltrates, demyelination, axonal injury and loss of neurological function1. Various cytokines which drive inflammatory responses are secreted by T cells and macrophages and are thought to be key mediators of autoimmune attack against CNS myelin. Elevated mRNA and protein levels of pro-inflammatory cytokines such as interleukin (IL)-6, interleukin-1β and tumor necrosis factor (TNF)-α, have been detected in MS lesions, cerebrospinal fluid and peripheral blood monocytes2. Interestingly, the activation of cannabinoid (CB) receptors 1 and 2 has profound effects, mostly inhibitory, on cytokine production3 and strongly appears to reduce symptoms associated with MS4. Although these inhibitory actions are thought to be mediated through neuro- and immunoregulatory mechanisms, few have investigated the regulation of these receptors in MS. Hence, this study first examined the expression level of these two receptors and compared it to that of pro-inflammatory cytokines in MS blood. Secondly, hypothesizing that cytokine-increase in MS enhances CB1 and CB2 mRNA, we investigated the effects of cytokines on CB1 and CB2 receptor expression in blood.

RNA was extracted from whole blood donated by healthy (n=38: 26 females; aged 27-62) and MS (n=44: 29 females; 15 males; 6 secondary progressive MS; 38 relapsing-remitting MS; aged 28-64; EDSS*2.0-7.5) subjects. CB1, CB2, IL-6, IL-1β, and TNFα mRNA from each sample was then measured and compared by quantitative reverse transcriptase (QRT)-PCR. Whole blood collected from healthy subjects (n=10) were stimulated with the three pro-inflammatory cytokines [IL-6, IL-1β 100ng/ml; TNFα 25ng/ml] and incubated for 18hrs at 37° C (5% CO2). RNA extraction, QRT-PCR and protein analysis using flow cytometric indirect staining methods for CB1 and CB2 expression were also performed. CB receptor and cytokine mRNA was found to be significantly up-regulated in MS blood when compared to that of normal subjects (CB1= 4-fold; CB2=5.1-fold; IL-6= 17-fold; IL-1β= 5.2-fold; TNFα= 6.3-fold; SEM<0.30; p= 0.001-0.027). Cytokine-stimulated whole blood also showed elevated CB1 (2-fold (IL-6); 1.8-fold (IL-1β); 3.4-fold (TNFα); SEM< 0.20; p= 0.016 for TNFα) and CB2 (2.6-fold (IL-6); 2.2-fold (IL-1β); 5.2-fold (TNFα); SEM<0.20; p= 0.02 for TNFα) mRNA and protein levels in CB positive immunoreactivity (TNFα-induced CB1=2.3±0.59%; CB2=3.7±0.96%) as opposed to non-stimulated samples. The parallel up-regulation of pro-inflammatory cytokines and cannabinoid receptors in MS blood suggests a role for cytokines not only in the pathogenesis of MS but also in the regulation of cannabinoid receptors. Such regulation may be induced as a protective mechanism against pro-inflammatory cytokine actions which are known to have detrimental effects in MS.

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* Expanded Disability Status Scale (Kurtzke J.F. (1983) Neurology 33, 1444-1452